The immune system is one of the most complex of the body's many intricate systems. A vast and complicated arrangement made up of many different types of cells and involving many different kinds of molecules, the human immune system allows the body to respond to foreign invaders such as bacteria, viruses, and other infectious agents, as well as foreign material such as pollen. In general, the human immune system is divided into two main parts, antibody-mediated immunity (also called “humoral” or “circulating” immunity) and cell-mediated immunity, both of which are managed by lymphocytes. Lymphocytes are one of the five kinds of white blood cells (leukocytes) circulating in the blood. There are several kinds of lymphocytes, each with different functions to perform. The most common types of lymphocytes are B lymphocytes (B cells), which are responsible for making antibodies, and T lymphocytes (T cells). Cells of the immune system not only include T cells and B cells, but also Natural Killer Cells, granulocytes (or polymorphonuclear (PMN) leukocytes), macrophages, and dendritic cells. The humoral system is managed by B cells with help from T cells and deals with infectious agents in the blood and tissues of the body. The cell-mediated system is managed by T cells and deals with cells of the body that have been infected.
An antigen is a substance, usually macromolecular, that stimulates or induces an immune response. Because of its complex macromolecular structure, a single microorganism consists of multiple antigens (e.g., surface structures such as cell wall components, fimbriae, flagella, etc., or extracellular proteins, such as toxins or enzymes produced by the microorganism). The coat proteins and some of the envelope proteins of animal viruses are also usually antigenic. A host is generally able to respond specifically to antigens that come into contact with components of its immune system. Both the antibody-mediated immunity and cell-mediated immunity systems involve complex interrelationships that allow them to mount immune reactions to almost any antigen. In other words, the immune system is able to recognize foreign substances (antigens) that stimulate the system to produce antibody-mediated immunity, cell-mediated immunity, or both.
The immune system complex is constituted by a variety of different cell types and organs disseminated throughout the body. These include the primary lymphoid organs, i.e., the bone marrow and the thymus. All the cells of the immune system are initially derived from the bone marrow in a process called hematopoiesis. During hematopoiesis bone marrow-derived stem cells differentiate into either mature cells of the immune system (“B” cells) or into precursors of cells that migrate out of the bone marrow to mature in the thymus (“T” cells). In addition to red blood cells, platelets, and B cells, the bone marrow also produces Natural Killer cells, granulocytes, and immature thymocytes. The function of the thymus is to produce mature T cells. Immature thymocytes, also known as prothymocytes, leave the bone marrow and migrate into the thymus where they mature and are then released into the bloodstream
The immune system complex also includes secondary lymphoid organs, e.g., the spleen, the lymph nodes, etc., as well as a circulatory system that is separate from blood vessels. The spleen, made up of B cells, T cells, macrophages, dendritic cells, Natural Killer cells, and red blood cells, is an immunologic filter of the blood. In addition to capturing antigens from the blood that passes through the spleen, migratory macrophages and dendritic cells bring antigens to the spleen via the bloodstream. An immune response is initiated in the spleen when macrophages or dendritic cells present the antigen to the appropriate B or T cells, and B cells become activated and produce large amounts of antibody. Lymphatic vessels and lymph nodes are the parts of a special circulatory system that carries lymph, a transparent fluid containing white blood cells, chiefly lymphocytes. Lymph bathes the tissues of the body, and is then collected in lymphatic vessels. Lymph nodes dot the network of lymphatic vessels and function as an immunologic filter for lymph when afferent lymph ducts bring lymph-containing antigens into the node. Composed mostly of T cells, B cells, dendritic cells, and macrophages, the lymph nodes drain fluid from most tissues. Antigens are filtered out of the lymph in the lymph node before the lymph is returned to the circulation. Macrophages and dendritic cells that capture antigens also present these foreign materials to T and B cells in the lymph nodes, resulting in the stimulation of B cells to develop there into antibody-secreting plasma cells. Antibodies leave the lymph node by the efferent ducts that empty into the blood stream. Lymphocytes can also leave the node by the efferent duct and travel to other sites in the lymphatic system or enter into the blood circulation. A single lymphocyte completes a circuit through the circulating blood and lymphatic systems once every 24 hours.
Tonsils, adenoids, Peyer's patches, and the appendix are also lymphoid tissues. Peyer's patches (masses of lymphocytes) are similar to the tonsils and are found throughout the body, especially in the mucous linings of the digestive and respiratory tracts. It is the function of the phagocytic cells found in Peyer's patches and other lymphatic aggregate follicles to defend the body against, for example, inadequately digested food particles crossing the gut wall and entering the blood, and to attack unwanted foreign invaders while they are still in the bowel.
The major function of B cells is the production of antibodies in response to foreign proteins of bacteria, viruses, and tumor cells. T cells are usually divided into two major groups, namely, the cytotoxic T lymphcytes (“Tc” cells or CTLs) and the helper T cells (“Th” cells or T helper cells). Th cells, also referred to as CD4+ T cells, function to augment or potentiate immune responses by the secretion of specialized factors that activate other white blood cells to fight off infection. They enhance the production of antibodies by B cells. Tc cells, also called CD8+ T cells, can directly kill certain tumor cells, viral-infected cells, and sometimes parasites. Tc cells are also important in down-regulation of immune responses. Both types of T cells often depend on the secondary lymphoid organs (the lymph nodes and spleen) as sites where activation occurs, but they are also found in other tissues of the body, including the liver, lung, blood, and intestinal and reproductive tracts.
Natural Killer cells, often referred to as NK cells, represent another type of lymphocyte and are similar to the Tc cell subset. They function as effector cells that directly kill certain tumors such as melanomas and lymphomas, and viral-infected cells. They are called “natural” killers because, unlike cytotoxic T cells, they do not need to recognize a specific antigen before carrying out their function. While NK cells, unlike the Tc cells, kill their targets without prior activation in the lymphoid organs, NK cells activated by Th cell secretions will kill tumor or viral-infected targets more effectively. NK cells target tumor cells and protect against a wide variety of infectious microbes. In several immunodeficiency diseases, including AIDS, Natural Killer cell function is abnormal. Natural Killer cells may also contribute to immunoregulation by secreting high levels of influential lymphokines. Some NK cells have surface receptors (FcγRIII, also called CD16) for the Fc portion of the IgG antibody. They bind to target cells through receptors for the Fc portion of an antibody that has reacted with antigen on a target cell. This type of cell-mediated immunity is called antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells may also have receptors for the C3 component of complement, another immune defense system, and therefore recognize cells that are coated with C3 as targets. ADCC is thought to be an important defense against a variety of parasitic infections caused, for example, by protozoa and helminths.
Although small lymphocytes look identical, they can be distinguished by molecules carried on their cell surface. Not only do such markers distinguish between B cells and T cells, they distinguish among various subsets of cells that behave differently. Every mature T cell, for instance, carries a marker known as T3 (or CD3). In addition, most helper T cells carry a T4 (CD4) marker, a molecule that recognizes Class II major histocompatibility complex (“MHC”) antigens. A molecule known as T8 (CD8), which recognizes Class I MHC antigens, is found on many suppressor/cytotoxic T cells.
Another group of white blood cells collectively referred to as granulocytes, or polymorphonuclear leukocytes (PMNs), is composed of three cell types. These cells, neutrophils, eosinophils, and basophils are important in the removal of bacteria and parasites from the body. Neutrophils migrate through capillary walls and into infected tissue where they kill invaders (e.g., bacteria) and then engulf the remnants by phagocytosis. Eosinophils are cytotoxic, releasing the contents of their granules on an invader. Basophils leave the blood and accumulate at the site of an infection or other inflammation and discharge the contents of their granules, releasing a variety of mediators such as histamine, serotonin, prostaglandins and leukotrienes that, for example, increase blood flow to the area. Mediators released by basophils also play an important part in some allergic responses such as hay fever and anaphylactic responses to insect stings.
Monocytes are large phagocytic white blood cells released from the bone marrow into the blood circulation. When a monocyte enters tissue, it develops into a macrophage. Macrophages are also large, phagocytic cells that engulf foreign material (antigens) that enter the body, as well as dead and dying cells of the body. Macrophages are important in the regulation of immune responses, and are often referred to as scavengers, or antigen-presenting cells (APCs) because they pick up and ingest foreign materials and present these antigens to other cells of the immune system such as T cells and B cells. This is one of the important first steps in the initiation of an immune response. Stimulated macrophages exhibit increased levels of phagocytosis and also secrete Interleukin-1 (IL-1), a product that helps to activate B cells and T cells.
Dendritic cells also originate in the bone marrow and function as APCs. They are usually found in the structural compartment of lymphoid organs such as the thymus, lymph nodes and spleen, but are also found in the bloodstream and other tissues. It is believed that dendritic cells capture antigen or bring it to the lymphoid organs where an immune response is initiated.
Important features of the immunological system relevant to host defense and/or immunity to pathogenic microorganisms include specificity, memory, and tolerance. It is understood, for example, that an antibody or reactive T cell will react specifically with the antigen that induced its formation; it will not react with other antigens. Generally, this specificity is of the same order as that of enzyme-substrate specificity or receptor-ligand specificity, although cross-reactivity is possible. The specificity of the immune response is explained by clonal selection. During the primary immune response, a specific antigen selects a pre-existing clone of specific lymphocytes and stimulates its activation, proliferation and differentiation. It is also understood that once the immune system has responded to produce a specific type of antibody or reactive T cell, it is capable of producing more of the antibody or activated T cell more rapidly and in larger amounts; this is called the secondary (or memory) response. It is also recognized that an animal generally does not undergo an immunological response to its own (potentially-antigenic) components. The animal is said to be tolerant, or unable to react to its own potentially antigenic components. This ensures that under normal conditions, an immune response to “self” antigens (called an autoimmune response) does not occur. Tolerance is brought about in a number of ways, but in essence the immune system is able to distinguish “self” components from “non-self” (foreign) antigens; it will respond to “non-self” but not to “self”. Sometimes in an animal, tolerance can be “broken”, which may result in an autoimmune disease.
The biological activities of the antibody-mediated and cell-mediated immune responses are different and vary from one type of infection to another. There are several classes or types of antibodies (and subclasses of various types) involved in antibody-mediated immunity. All of the classes of antibodies that are produced in response to a specific antigen react stereochemically with that antigen and not with other (different) antigens. The host has the genetic capacity to produce specific antibodies to thousands of different antigens, but does not do so until there is an appropriate (specific) antigenic stimulus. Due to clonal selection, the host produces only the homologous antibodies that will react with that antigen which, as noted above, are found in blood (plasma), lymph, and many extravascular tissues. Once the antibody-mediated immunity response occurs following interaction of B lymphocytes with antigen and their differentiation into antibody-secreting plasma cells, the secreted antibody binds to the antigen which, in turn, results in its neutralization or elimination from the body.
Cell-mediated immunity, on the other hand, is mediated by specific subpopulations of T-lymphocytes called effector T cells that exist in precursor form as “resting T cells” (pT cells). These cells bear receptors for specific antigens and recognize these antigens on the surfaces of other cells. Stimulation with that antigen results in T cell activation. T cells enlarge, enter into a mitotic cycle, reproduce and develop into effector T cells whose activities are responsible for this type of immunity. They also develop into clones of identical reactive T cells called memory T cells. As noted above, most of the T cells in the body belong to one of two subsets and are distinguished by the presence on their surface of one or the other of two glycoproteins designated CD4 and CD8. Which of these molecules is present determines the types of cells to which the T cell can bind. T cells bearing CD8 (CD8+ T cells) always recognize antigen in association with Class I MHC proteins and typically function as cytotoxic T cells. Almost all the cells of the body express Class I MHC molecules. T cells bearing CD4 (CD4+ T cells) always recognize antigens in association with Class II MHC proteins on the surfaces of other cells. Only specialized antigen-presenting cells express Class II MHC molecules, including dendritic cells, phagocytic cells such as macrophages, and B cells. CD4+ T lymphocytes generally function as T helper cells.
T helper cells, which include Th1 cells and Th2 cells, respond to antigen with the production of lymphokines. Th1 and Th2 cells can be distinguished based on their lymphokine profiles. Like all T cells, Th cells arise in the thymus. When they are presented with an antigen by antigen-presenting dendritic cells they begin to proliferate and become activated. There are two kinds of dendritic cell, DC1 cells (descended from monocytes) and DC2 cells (which appear to be derived from lymphocytes).
Th1 cells (inflammatory Th1 cells involved in the elimination of pathogens residing intracellularly in vesicular compartments) are produced when DC1-type dendritic cells present antigen to the T cell receptor for antigen (TCR) and secrete Interleukin 12 (IL-12). This paracrine stimulation activates Th1 cells to secrete their own lymphokines, in particular, Tumor-Necrosis Factor-beta (TNF-β) (also known as lymphotoxin) and interferon-gamma (IFN-γ). These lymphokines stimulate macrophages to kill bacteria they have engulfed by phagocytosis and they recruit other leukocytes to the site producing inflammation. Th1 cells are essential for cell-mediated immunity and for controlling intracellular pathogens such as, for example, Listeria and Mycobacterium tuberculosis. 
Th2 cells (“true” helper Th2 cells, which are required for antibody production by B cells) are produced when DC2-type dendritic cells present antigen to the T cell receptor for antigen and, presumably, one or more paracrine stimulants. The major lymphokines secreted by Th2 cells are Interleukin 4 (IL-4), which stimulates class-switching in B cells and promotes their synthesis of IgE antibodies, acts as a positive-feedback device promoting more pre-Th cells to enter the Th2 pathway, and blocks expression of the IL-12 receptor thereby inhibiting pre-Th cells in the thymus from entering the Th1 pathway. IL-4 also causes B cells to proliferate and differentiate into antibody-secreting plasma cells and memory B cells. IL-4 activates only B cells in the vicinity which themselves have bound the antigen, and not others, so as to sustain the specificity of the immune response. Th2 cells also produce Interleukin 5 (IL-5, which attracts and activates eosinophils), Interleukin 10 (IL-10, which inhibits IL-12 production by DCs and prevents maturation of pre-Th cells to Th1 cells), and Interleukin 13 (IL-13, which also promotes the synthesis of IgE antibodies).
Activation of the Th2 cell also causes it to begin to produce Interleukin 2 (IL-2), and to express a membrane receptor for IL-2. The secreted IL-2 autostimulates proliferation of Th2 cells. For example, IL-2 binds IL-2 receptors on other T cells (which have bound the antigen) and stimulates their proliferation. In addition IL-2, stimulated Th2 cells also produce IFN-γ and Interleukin 6 (IL-6), which mediate various aspects of the immune response. IFN-β activates Natural Killer cells to their full cytolytic potential, and is an activator of macrophages and thus increases their antitumor activities. If the macrophages are infected by intracellular parasites, it activates macrophages which in turn destroy the parasites. IFN-γ.quadrature. also reinforces the antitumor activities of the cytotoxic lymphocytes, increases the nonspecific activities of NK-cells, and is one of the factors that controls the differentiation of B cells and increases the secretion of immunoglobins. IL-6 stimulates several types of leukocytes, as well as the production of Acute Phase Proteins in the liver. It is particularly important in inducing B cells to differentiate into antibody forming (plasma) cells. Thus, Th2 cells provide help for B cells and are essential for antibody-mediated immunity.
Cytotoxic T lymphocytes are able to kill cells that show a new or foreign antigen on their surface (for example, virus-infected cells, or tumor cells, or transplanted tissue cells). The CD8+ CTLs also come in two subsets: Tc1 that, like Th1 cells, secrete IFN-γ, and Tc2 that, like Th2 cells, secrete IL-4.
The cell-mediated immunity response also plays a role in destruction of tumor cells and in rejection of tissue transplants in animals. A major problem in tissue transplantation is rejection, which is often based on cell-mediated immunity response to “foreign” cells (because they are not a perfect antigenic match). Because tumor cells contain specific antigens not seen on normal cells they also may be recognized as foreign and destroyed by the forces of cell-mediated immunity. If tumor cells develop on a regular basis in animals, it may be cell-mediated immunity that eliminates them or holds them in check. The increase in the incidence of many types of cancer (tumors) in humans with advancement of age may be correlated with a decline in the peak efficiency of the immune system that occurs about 25 years of age.
A summary of the types of cells involved in the expression of cell-mediated immunity follows. Tc lymphocytes kill cells bearing foreign antigen on surface in association with Class I MHC and can kill cells that are harboring intracellular parasites (either bacteria or viruses) as long as the infected cell is displaying a microbial antigen on its surface. Tc cells kill tumor cells and account for rejection of transplanted cells. Tc cells recognize antigen-Class I MHC complexes on target cells, contact them, and release the contents of granules directly into the target cell membrane which lyses the cell. Th lymphocytes produce lymphokines that are “helper” factors for development of B cells into antibody-secreting plasma cells. They also produce certain lymphokines that stimulate the differentiation of effector T lymphocytes and the activity of macrophages. Th1 cells recognize antigen on macrophages in association with Class II MHC and become activated (by IL-1) to produce lymphokines including IFN-γ that activates macrophages and NK cells. These cells mediate various aspects of the cell-mediated immunity response including delayed-type hypersensitivity reactions. Th2 cells recognize antigen in association with Class II MHC on an APC and then produce interleukins and other substances that stimulate specific B cell and T cell proliferation and activity. Macrophages are important as antigen-presenting cells that initiate T cell interactions, development, and proliferation. Macrophages are also involved in expression of cell-mediated immunity because they become activated by IFN-γ produced in a cell-mediated immunity response. Activated macrophages have increased phagocytic potential and release soluble substances that cause inflammation and destroy many bacteria and other cells. Natural Killer cells are cytotoxic cells that lyse cells bearing new antigen regardless of their MHC type and even lyse some cells that bear no MHC proteins. NK cells are defined by their ability to kill cells displaying a foreign antigen (e.g., tumor cells) regardless of MHC type and regardless of previous sensitization (exposure) to the antigen. NK cells can be activated by IL-2 and IFN-γ, and lyse cells in the same manner as cytotoxic T lymphocytes. Some NK cells have receptors for the Fc domain of the IgG antibody and are thus able to bind to the Fc portion of IgG on the surface of a target cell and release cytolytic components that kill the target cell via antibody-dependent cell-mediated cytotoxicity.
Extracellular factors that affect cell proliferation and differentiation have been defined as cytokines. These include the lymphokines, which are proteins produced by T-lymphocytes that have effects on the differentiation, proliferation, and activity of various cells involved in the expression of cell-mediated immunity. In general, lymphokines function by (1) focusing circulating leukocytes and lymphocytes into the site of immunological encounter; (2) stimulating the development and proliferation of B cells and T cells; (3) stimulating and preparing macrophages for their phagocytic tasks; (4) stimulating Natural Killer cells; and (5) providing antiviral cover and activity. A summary of various important lymphokines follows. Initially referred to as lymphocyte activation factor, IL-1 is mainly a product of macrophages, and has a variety of effects on various types of cells. It acts as a growth regulator of T cells and B cells, and it induces other cells such as hepatocytes to produce proteins relevant to host defense. IL-1 forms a chemotactic gradient for neutrophils and serves as an endogenous pyrogen which produces fever. Thus, IL-1 plays an important role in both the immune responses and in the inflammatory response. IL-2 stimulates the proliferation of T cells and activates NK cells. IL-3 regulates the proliferation of stem cells and the differentiation of mast cells. IL-4 causes B cell proliferation and enhanced antibody synthesis. IL-6 (also referred to as Interferon-beta2, hybridoma growth factor, B-cell differentiation factor, and hepatocyte stimulatory factor) has effects on B cell differentiation and on antibody production and on T cell activation, growth, and differentiation, and probably has a major role in the mediation of the inflammatory and immune responses initiated by infection or injury. IL-8 is a chemotactic attractant for neutrophils. IL-13 shares many of the properties of IL-4, and is a potent regulator of inflammatory and immune responses. IFN-γ is produced by T cells and may be considered a lymphokine. It is sometimes called “immune interferon” (Interferon-alpha being referred to as “leukocyte interferon” and Interferon-beta being referred to as “fibroblast interferon”). IFN-γ has several antiviral effects including inhibition of viral protein synthesis in infected cells. It also activates macrophages and NK cells, and stimulates IL-1, IL-2, and antibody production. Lymphotoxins include the Tumor Necrosis Factors. TNF-beta is produced by T cells, while TNF-alpha is produced by T cells as well as other types of cells. TNFs function to kill cells, including tumor cells (at a distance). There are several Colony Stimulating Factors (CSFs), including granulocyte macrophage colony stimulating factor (GMCSF), which cause phagocytic white cells of all types to differentiate and divide.
The nature of the membrane receptors for antigen on B cells and T cells is fairly well understood. Each B cell has approximately 105 membrane-bound antibody molecules (IgD or IgM) that correspond in specificity to the antibody the cell is programmed to produce (these receptors being referred to as BCRs). CD32 (FcγRII) on B cells are receptors for the Fc region of IgG. CD21 and CD35 on B cells are receptors for complement components. Each T cell has about 105 molecules of a specific antigen-binding T cell receptor (a TCR) exposed on its surface. The TCR is similar, but not identical, to an antibody. There are two types of T cells that differ in their TCRs, alpha/beta (αβ) T cells and gamma/delta (γδ) T cells. The TCR of alpha/beta T cells binds a bimolecular complex displayed by a Class I MHC molecule at the surface of an antigen-presenting cell. As noted above, most Th cells express CD4, whereas most Tc cells express CD8.
Both BCRs and TCRs are similar in that they are integral membrane proteins, they are present in thousands of identical copies exposed at the cell surface, they are made before the cell ever encounters an antigen, they are encoded by genes assembled by the recombination of segments of DNA, they have a unique binding site that binds through non-covalent forces to a portion of the antigen called an epitope (or antigenic determinant) that depends on complementarity of the surface of the receptor and the surface of the epitope, and successful binding of the antigen receptor to the epitope, if accompanied by additional signals, results in stimulation of the cell to leave G0 and enter the cell cycle and repeated mitosis that leads to the development of a clone of cells bearing the same antigen receptor, i.e., a clone of cells of the identical specificity. BCRs and TCRs differ in their structure, the genes that encode them, and the type of epitope to which they bind.
Induction of a primary immune response begins when an antigen penetrates epithelial surfaces. It will eventually come into contact with macrophages or certain other classes of antigen presenting cells, including B cells, monocytes, dendritic cells, Langerhans cells, and endothelial cells. Antigens, such as bacterial cells, are internalized by endocytosis and “processed” by APCs, then “presented” to immunocompetent lymphocytes to initiate the early steps of the immunological response. Processing by a macrophage (for example) results in attaching antigenic materials to the surface of the membrane in association with Class II MHC molecules on the surface of the cell. The antigen-class II MHC complex is presented to a T-helper (Th2) cell which is able to recognize processed antigen associated with a Class II MHC molecule on the membrane of the macrophage. This interaction, together with stimulation by IL-1 from secreted by the macrophage, will activate the Th2 cell.
As indicated above, B cells themselves behave as APCs. Cross-linked antigens bound to antibody receptors on the surface of a B cell cause internalization of some of the antigen and expression on the B cell membrane together with Class II MHC molecules. The Th2 cell recognizes the antigen together with the Class II MHC molecules, and secretes the various lymphokines that activate the B cells to become antibody-secreting plasma cells and memory B cells. Even if the antigen cannot cross-link the receptor, it may be endocytosed by the B cell, processed, and returned to the surface in association with Class II MHC where it can be recognized by specific Th2 cells which will become activated to initiate B cell differentiation and proliferation. In any case, the overall B cell response leads to antibody-mediated immunity.
The antigen receptors on B cell surfaces are thought to be the specific types of antibodies that they are genetically-programmed to produce. Hence, there are thousands of sub-populations of B cells distinguished by their ability to produce a unique antibody molecule. B cells can also react with a homologous antigen on the surface of the macrophage, or with soluble antigens. When a B cell is bound to antigen, and simultaneously is stimulated by IL-4 produced by a nearby Th2 cell, the B cell is stimulated to grow and divide to form a clone of identical B cells, each capable of producing identical antibody molecules. The activated B cells further differentiate into plasma cells which synthesize and secrete large amounts of antibody, and into memory B cells. The antibodies produced and secreted by the plasma cells will react specifically with the homologous antigen that induced their formation. Many of these reactions lead to host defense and to prevention of reinfection by pathogens. Memory cells play a role in secondary immune responses. Plasma cells are relatively short-lived (about one week) but produce large amounts of antibody during this period. Memory cells, on the other hand, are relatively long-lived and upon subsequent exposure to antigen they become quickly transformed into antibody-producing plasma cells.
Generation of cell mediated immunity begins when, for example, a cytotoxic T cell recognizes a processed antigen associated with Class I MHC on the membrane of a cell (usually an altered self cell, but possibly a transplanted tissue cell, or a eukaryotic parasite). Under stimulation by IL-2 produced by Th2 cells, the Tc cell becomes activated to become a cytotoxic T lymphocyte capable of lysing the cell which is showing the new foreign antigen on its surface, a primary manifestation of cell-mediated immunity. The interaction between an antigen-presenting macrophage and a Th cell stimulates the macrophage to produce and secrete a Interleukin-1 that acts locally on the Th cell, stimulating the Th-cell to differentiate and produce its own cytokines (which may here be called lymphokines because they arise from a lymphocyte). These lymphokines have various functions. IL-4 has an immediate effect on nearby B cells. IL-2 has an immediate effect on T cells as described above.
Leucocytes also express adhesion promoting receptors that mediate cell-cell and cell-matrix interactions. These adhesive interactions are crucial to the regulation of haemopoiesis and thymocyte maturation, the direction and control of leucocyte traffic and migration through tissues, and the development of immune and non-immune inflammatory responses. Several families of adhesion receptors have been identified. The leucocyte integrin family comprises three alpha-beta heterodimeric membrane glycoproteins that share a common beta subunit, designated CD18. The alpha subunits of each of the three members, lymphocyte function associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1) and p150,95 are designated CD11a, b, and c respectively. These adhesion molecules play a critical part in the immune and inflammatory responses of leucocytes. The leucocyte integrin family is, in turn, part of the integrin superfamily, members of which are evolutionally, structurally and functionally related. Another Integrin subfamily found on leucocytes is the VLA group, so-called because the “very late activation antigens” VLA-1 and VLA-2 were originally found to appear late in T-cell activation. Members of this family function mainly as extracellular matrix adhesion receptors and are found both on haemopoietic and non-haemopoietic cells. They play a part in diverse cellular functions including tissue organisation, lymphocyte recirculation and T-cell immune responses. Another integrin subfamily, the cytoadhesins, are receptors on platelets and endothelial cells that bind extracellular matrix proteins. A second family of adhesion receptors is the immunoglobulin superfamily, members of which include CD2, LFA-3, and ICAM-1, which participate in T-cell adhesive interactions, and the antigen-specific receptors of T and B cells, CD4, CD8, and the MHC Class I and II molecules. Another recognized family of adhesion receptors is the selectins, characterized by a common lectin domain. Leucocyte adhesion molecule-1 (LAM-1), which is the human homologue of the murine homing receptor, MEL-14, is expressed on leucocytes, while endothelial leucocyte adhesion molecule-1 (ELAM-1) and granule membrane protein (GMP-140) are expressed on stimulated endothelial cells and activated platelets.
Activation of an immune response requires physical cell-cell contact in addition to cytokines. Thus, for example, development of B and T cell precursors require intimate contact with stromal cells. At least three critical cell-cell contact events are required for the generation of immune responses. The first is initial contact of a specific antigen with a naive T cell. Because of the requirement for MHC presentation, this is an obligate cell contact event. In normal situations the critical antigen presenting cell is the dendritic cell. In addition to the MHC/peptide-TCR interaction there are other non-antigen specific membrane bound ligand-receptor pairs which are important for the dendritic cell-T cell interaction. The principal one is the association of the CD28 molecule on the T cell with either of two ligands, B7.1 (CD80) and B7.2 (CD86), on the dendritic cell. These molecules are termed accessory molecules and it is understood that the CD28 molecule delivers an essential second signal to the T cell without which the T cell does not become activated.
A second essential cell-cell contact is between the activated T cell and an antigen-specific B cell. Most antigens are T cell-dependent, that is, an antibody response to the antigen absolutely requires T cell help. This help is delivered both by cytokines and by cell-cell contact. Cells bind specific antigen via surface Ig, then internalize, process, and present it on Class II MHC molecules. This enables them to be recognized by T cells specific for helper epitopes from the specific antigen. This cell-cell interaction also requires CD28 binding to B7 on the B cell. In addition, a molecule called CD40 ligand or CD154, the expression of which is induced upon T cell activation, binds to CD40 on B cells. CD40 crosslinking promotes B cell proliferation, prevents apoptosis of germinal-center B cells, and promotes immunoglobulin isotype switching. The CD28-B7 and CD40-CD40L receptor ligand interactions are both essential for the dialogue between B and T cells that causes their mutual activation.
A third cell-cell interaction that is essential in immune responses is the binding of activated B cells (which have migrated into a specialised structure in lymphoid organs called germinal centers) to follicular dendritic cells (FDCs). FDCs are specialized stromal cells that hold intact, i.e., unprocessed, antigen on their surface in the form of long-lived immune complexes. Among other molecules, FDCs express CD23, which binds to germinal center B cells via a CR2 receptor and stimulates differentiation to plasma cells. Time is required before a primary immune response is effective as a host defense. Antigens have to be recognized, taken up, digested, processed, and presented by APCs. A few select Th cells must react with antigen and respond; preexisting B or T lymphocytes must encounter the antigen and proliferate and differentiate into effector cells (plasma cells or Tc cells). In the case of antibody-mediated immunity, antibody level has to build up to an effective physiological concentration to render its host resistant. It may take several days or weeks to reach a level of effective immunity, even though this immunity may persist for many months, or years, or even a lifetime, due to the presence of the antibodies. In natural infections, the inoculum is small, and even though the antigenic stimulus increases during microbial replication, only small amounts of antibody are formed within the first few days, and circulating antibody is not detectable until about a week after infection.
With regard to induction of a secondary immune response, it is understood that on re-exposure to microbial antigens (secondary exposure to antigen), there is an accelerated immunological response, i.e., the secondary or memory response. Larger amounts of antibodies are formed in only 1-2 days. This is due to the activities of specific memory B cells or memory T cells which were formed during the primary immune response. These memory cells, when stimulated by homologous antigen, “remember” having previously seen the antigen, and are able to rapidly divide and differentiate into effector cells. Stimulating memory cells to rapidly produce very high (effective) levels of persistent circulating antibodies is the basis for giving “booster”-type vaccinations to humans and pets. Thus, following the first exposure to an antigen the immune response (as evidenced by following the concentration of specific antibody in the serum) develops gradually over a period of days, reaches a low plateau within 2-3 weeks, and usually begins to decline in a relatively short period of time. When the antigen is encountered a second time, a secondary (memory) response causes a rapid rise in the concentration of antibody, reaching a much higher level in the serum, which may persist for a relatively long period of time. This is not to say that a protective level of antibody may not be reached by primary exposure alone, but usually to ensure a high level of protective antibody that persists over a long period of time, it is necessary to have repeated antigenic stimulation of the immune system.
An immunoglobulin molecule (abbreviated Ig), is a multimeric protein composed of two identical light chain polypeptides and two identical heavy chain polypeptides (H2L2) that are joined into a macromolecular complex by interchain disulfide bonds, i.e., covalent bonds between the sulfhydryl groups of neighboring cysteine residues. There are various classes of human antibody proteins, each of which is produced by a specific clone of plasma cells. Five human immunoglobulin classes are defined on the basis of their heavy chain composition, and are named IgG, IgM, IgA, IgE, and IgD. The IgG-class and IgA-class antibodies are further divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2. Intrachain disulfide bonds join different areas of the same polypeptide chain, which results in the formation of loops that, along with adjacent amino acids, constitute the immunoglobulin domains. At the amino-terminal portion (also called the “NE2-terminus” or the “N-terminus”), each light chain and each heavy chain has a single variable region that shows considerable variation in amino acid composition from one antibody to another. The light chain variable region, VL, associates with the variable region of a heavy chain, VH, to form the antigen binding site of the immunoglobulin, called the Fv.
In addition to variable regions, each of the antibody chains have one or more constant regions. Light chains have a single constant region domain. Thus, light chains have one variable region and one constant region. Heavy chains have several constant region domains. The heavy chains in IgG, IgA, and IgD antibodies have three constant region domains, which are designated CH1, CH2, and CH3, and the heavy chains in IgM and IgE antibodies have four constant region domains, CH1, CH2, CH3 and CH4. Thus, heavy chains have one variable region and three or four constant regions. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988).
The heavy chains of immunoglobulins can also be divided into three functional regions: the Fd region (a fragment comprising VH and CH1, i.e., the two N-terminal domains of the heavy chain), the hinge region, and the Fc region (the “fragment crystallizable” region, derived from constant regions and formed after pepsin digestion). The Fd region in combination with the light chain forms an Fab (the “fragment antigen-binding”). Because an antigen will react stereochemically with the antigen-binding region at the amino terminus of each Fab the IgG molecule is divalent, i.e., it can bind to two antigen molecules. The Fc contains the domains that interact with immunoglobulin receptors on cells and with the initial elements of the complement cascade. Thus, the Fc fragment is generally considered responsible for the effector functions of an immunoglobulin, such as complement fixation and binding to Fc receptors. Pepsin sometimes also cleaves before the third constant domain (CH3) of the heavy chain to give a large fragment F(abc) and a small fragment pFcb. These terms are also used for analogous regions of the other immunoglobulins. The hinge region, found in IgG, IgA, and IgD class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses.
For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, it is relatively short and contains a rigid poly-proline double helix, stabilised by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3 the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge region reportedly decreases in the order IgG3>IgG1>IgG4>IgG2. The four IgG subclasses also differ from each other with respect to their effector functions. This difference is related to differences in structure, including with respect to the interaction between the variable region, Fab fragments, and the constant Fc fragment. Nevertheless, aside from glycosylation within the CH2 region, for example, in spite of this knowledge there are no set rules or conventions regarding means or methods to change features, including sequences, of these subclasses of molecule to change, control, add, or remove different functions, for example, ADCC, CDC, and other functions.
According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. Shin et al., 1992 Immunological Reviews 130: 87. The upper hinge region includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 515) which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.
Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may also affect the effector functions of the Fc portion of the antibody. Three general categories of effector functions associated with the Fc region include (1) activation of the classical complement cascade, (2) interaction with effector cells, and (3) compartmentalization of immunoglobulins. The different human IgG subclasses vary in the relative efficacies with which they fix complement, or activate and amplify the steps of the complement cascade. See, e.g., Kirschfink, 2001 Immunol. Rev. 180: 177; Chakraborti et al., 2000 Cell Signal 12: 607; Kohl et al., 1999 Mol. Immunol. 36: 893; Marsh et al., 1999 Curr. Opin. Nephrol. Hypertens. 8: 557; Speth et al., 1999 Wien Klin. Wochenschr. 111: 378.
Complement-dependent cytotoxicity (CDC) is believed to be a significant mechanism for clearance of specific target cells such as tumor cells. CDC is a stream of events that consists of a series of enzymes that become activated by each other in a cascade fashion. Complement has an important role in clearing antigen, accomplished by its four major functions: (1) local vasodilation; (2) attraction of immune cells, especially phagocytes (chemotaxis); (3) tagging of foreign organisms for phagocytosis (opsonization); and (4) destruction of invading organisms by the membrane attack complex (MAC attack). The central molecule is the C3 protein. It is an enzyme that is split into two fragments by components of either the classical pathway or the alternative pathway. The classical pathway is induced by antibodies, especially IgG and IgM, while the alternative pathway is nonspecifically stimulated by bacterial products like lipopolysaccharide (LPS). Briefly, the products of the C3 split include a small peptide C3a which is chemotactic for phagocytic immune cells and results in local vasodilation by causing the release of C5a fragment from C5. The other part of C3, C3b coats antigens on the surface of foreign organisms and acts to opsonize the organism for destruction. C3b also reacts with other components of the complement system to form an MAC consisting of C5b, C6, C7, C8 and C9.
In general, IgG1 and IgG3 most effectively fix complement, IgG2 is less effective, and IgG4 does not activate complement. Complement activation is initiated by binding of C1q, a subunit of the first component C1 in the cascade, to an antigen-antibody complex. Even though the binding site for C1q is located in the CH2 domain of the antibody, the hinge region influences the ability of the antibody to activate the cascade. For example, recombinant immunoglobulins lacking a hinge region are unable to activate complement. Shin et al., 1992. Without the flexibility conferred by the hinge region, the Fab portion of the antibody bound to the antigen may not be able to adopt the conformation required to permit C1q to bind to CH2. See id. Hinge length and segmental flexibility have been reported to correlate with complement activation; however, the correlation is not absolute. Human IgG3 molecules with altered hinge regions that are as rigid as IgG4, for example, can still effectively activate the cascade.
The absence of a hinge region, or a lack of a functional hinge region, can also affect the ability of certain human IgG immunoglobulins to bind Fc receptors on immune effector cells. Binding of an immunoglobulin to an Fc receptor facilitates antibody-dependent cell-mediated cytotoxicity, which as noted above is presumed to be an important mechanism for the elimination of tumor cells. The human IgG Fc receptor (FcR) family is divided into three groups, FcγRI (CD64), which is capable of binding IgG with high affinity, and FcγRII (CD32) and FcγRIII (CD16), both of which are lower affinity receptors. The molecular interaction between each of the three receptors and an immunoglobulin has not been defined precisely, but experimental evidence indicates that residues in the hinge proximal region of the CH2 domain may be important to the specificity of the interaction between the antibody and the Fc receptor. IgG1 myeloma proteins and recombinant IgG3 chimeric antibodies that lack a hinge region are reportedly unable to bind FcγRI, perhaps because accessibility to CH2 is decreased. Shin et al., 1993 Intern. Rev. Immunol. 10: 177, 178-79.
Unusual and apparently evolutionarily unrelated exceptions to the H2L2 structure of conventional antibodies occur in some isotypes of the immunoglobulins found in camelids (camels, dromedaries and llamas; Hamers-Casteman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J. Mol. Biol. 275: 413), nurse sharks (Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in the spotted raffish (Nguyen, et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” 2002 Immunogenetics 54(1): 39-47). These antibodies can apparently form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as “heavy-chain antibodies” or “HCAbs”). In both species, these variable regions often contain an extended third complementarity determining region (CDR3) that may help compensate for the lack of a light chain variable region, and there are frequent disulfide bonds between CDR regions that presumably help to stabilize the binding site. Muyldermans et al., 1994 Prot. Engineer. 7: 1129; Roux et al., 1998. However, the precise function of the heavy chain-only “antibodies” is unknown, and the evolutionary pressure leading to their formation has not been identified. See, e.g., Nguyen, et al., 2002, supra. Camelids, including camels, llamas, and alpacas, also express conventional H2L2 antibodies, and the heavy chain-only antibodies thus do not appear to be present in these animals simply as an alternative antibody structure.
Variable regions (VHH) of the camelid heavy chain-only immunoglobulins and conventional (H2L2) heavy chain variable regions contain amino acid differences, including differences at several positions that may be involved in the interface between conventional VH and VL domains. Nguyen et al., 1998 J. Mol. Biol 275: 413; Muyldermans et al., 1994 Prot. Engineer. 7: 1129. Camelid VHH reportedly recombines with IgG2 and IgG3 constant regions that contain hinge, CH2, and CH3 domains and lack a CH1 domain. Hamers-Casterman et al., 1993 Nature 363: 446. Interestingly, VHH are encoded by a chromosomal locus distinct from the VH locus (Nguyen et al., 1998, supra), indicating that camelid B cells have evolved complex mechanisms of antigen recognition and differentiation. Thus, for example, llama IgG1 is a conventional (H2L2) antibody isotype in which VH recombines with a constant region that contains hinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that lack CH1 domains and that contain no light chains.
The classes of immunoglobulins have different physical and chemical characteristics and they exhibit unique biological properties. Their synthesis occurs at different stages and rates during an immune response and/or during the course of an infection. Their importance and functions in host resistance (immunity) are different.
Immunoglobulin G (IgG), a protein with a molecular weight of about 150,000 daltons (150 kD), is the predominant Ig in the serum. It makes up about 80% of the total antibody found in an animal at any given time, being 75% of the total serum antibody. It can diffuse out of the blood stream into the extravascular spaces and it is the most common Ig found there. Its concentration in tissue fluids is increased during inflammation, and it is particularly effective at the neutralization of bacterial extracellular toxins and viruses. It also has opsonizing ability and complement-fixing ability. The polypeptide composition, of the Fc region of all IgG1 antibody molecules is relatively constant regardless of antibody specificity; however, as noted above, the Fab regions always differ in their exact amino acid sequences depending upon their antigenic specificity. Specific amino acid regions of the Fc portion of the molecule are recognized by receptors on phagocytes and certain other cells, and the Fc domain contains a peptide region that will bind to and activate complement, which is often required for the manifestation of antibody-mediated immunity. Because the IgG molecule is divalent, it can cross-link antigen molecules, which may lead to precipitation or agglutination of antigens; if IgG is bound to antigen on a microbial cell or surface, its Fc region may provide an extrinsic ligand that will be recognized by specific receptors on phagocytes. Microbial cells or viruses coated with IgG molecules are opsonized for phagocytosis, and opsonized pathogens are taken up and destroyed much more readily by phagocytes than their non-opsonized counterparts. IgG, as well as IgM and IgA, will neutralize the activity of toxins, including bacterial exotoxins. Furthermore, cross-linked IgG molecules on the surface of a cell can bind and activate complement from the serum and set off a cascade of reactions that can lead to destruction of the cell.
IgM is the first immunoglobulin to appear in the blood stream during the course of an infection. It is mainly confined to the bloodstream and provides protection against blood-borne pathogens. IgM makes up about 10% serum immunoglobulins, and is arranged to resemble a pentamer of five immunoglobulin molecules (having a molecular weight of about 900 kD) tethered together at by their Fc domains. In addition to covalent linkages between the monomeric subunits, the pentamer is stabilized by a 1 Skd polypeptide called J chain. IgM, therefore, has a theoretical “valence” of ten (i.e., it has ten exposed Fab domains). Probably, the most important role of IgM is its ability to function early in the immune responses against blood-borne pathogens given its efficiency in agglutinating particulate antigens. IgM binds also complement strongly and IgM antibodies bound to a microbial surface act as opsonins, rendering the microbe more susceptible to phagocytosis. In the presence of complement and IgM whole microbial cells may be killed and lysed. As noted above, IgM also appears on the surfaces of mature B cells as a transmembranous monomer where it functions as an antigen receptor, capable of activating B cells when bound to antigen.
Gene rearrangement at the immunoglobulin loci during lymphoid development generates a repertoire of B lymphocytes that express a diversity of antigen receptors. The gene rearrangement, which is catalysed by the rearrangement-activating gene (“RAG”) recombinase, integrates the immunoglobulin V, D and J gene segments to yield productively rearranged immunoglobulin genes that encode the heavy and light chains of IgM antibodies. The diversity of IgM antibodies in this primary repertoire is achieved through combinatorial mechanisms (the choice of V, D and J gene segments utilized in a particular antibody), as well as junctional diversity. The joining of V, D and J gene segments is somewhat imprecise, and nucleotides may be inserted at the junction in a non-templated manner. There is therefore a very high degree of resultant diversity at the V-D-J borders. This contributes in a major way to the structural diversity of the third complementarity determining region of the antibody, a region that often plays a critical role in antigen recognition. This primary repertoire of IgM antibodies comprises a few million different structures. The size of this repertoire means that any incoming antigen is likely to encounter an antibody that recognizes it with acceptable affinity. A high-affinity binding site is unlikely to be available for most incoming antigens (the repertoire is not large enough), but the affinity of the available IgM antibodies in the primary repertoire will vary from antigen to antigen. If an epitope is re-iterated at high density on the surface of the antigen (e.g., a repeated structure on the surface of a virus or bacterium), then an IgM antibody may nevertheless be effective in mediating clearance of the organism, despite the low affinity of the individual interaction between antigenic epitope and immunoglobulin combining site. The density of the epitopes may allow multivalent interactions with IgM, leading to a high-avidity interaction, providing that a suitable spacing of antigenic epitopes can occur. Nevertheless, to ensure an effective and specific response, especially when the concentration of antigen is low (as may occur when the body is faced with a very small number of infecting viral particles), it would be preferable if high-affinity antibodies were available for neutralizing, for example, an infecting organism. The size of the primary repertoire mitigates against the likelihood of such high-affinity antibodies being present in this repertoire. The immune system therefore operates using a two-stage strategy. The primary repertoire of IgM antibodies is generated by a process of gene rearrangement and takes place prior to antigen encounter during early lymphocyte development. However, once foreign antigen has been encountered, those B cells in the primary repertoire that encode suitable (albeit low-affinity) antibodies are selectively expanded and subjected to an iterative alternation of directed hypermutation and antigen-mediated selection. This allows a significant maturation in affinity of the antigen-specific antibodies that are produced. Antigen triggering also drives isotype switch recombination. Thus, in the absence of external antigen stimulation and any maternally derived immunoglobulin, the serum will only contain a diversity of unmutated IgM molecules that have been generated by gene rearrangement. This repertoire shifts with age as a result of continuous antigen exposure, such that the majority of the serum immunoglobulin in older animals is composed of mutated IgG (and IgA) molecules whose specificities have developed as a consequence of antigen selection.
IgA exists as a H2L2 monomer of about 160 kD in serum and, in secretions, as a dimer of the H2L2 monomer of about 400 kD. As with IgM, polymerization (dimerization) is via a J-chain. IgA has two subclasses based on different heavy chains, IgA1 and IgA2. IgA1 is produced in bone marrow and makes up most of the serum IgA. Both IgA1 and IgA2 are synthesized in GALT (gut associated lymphoid tissues) to be secreted onto the mucosal surfaces. Because IgA may be synthesized locally and secreted in the seromucous secretions of the body, it is sometimes referred to as secretory antibody or sIgA. Quantitatively, IgA is synthesized in amounts greater than IgG, but it has a short half life in serum (6 days), and it is lost in secretory products. The concentration of IgA in serum is about 15% of the total antibody. Secretion of dimeric IgA is mediated by a 100 kD glycoprotein called secretory component. It is the addition of the secretory piece to IgA molecules that accounts for their ability to exit the body to mucosal surfaces via the exocrine glands. IgM can be transported similarly and makes up a small proportion of secretory antibodies. Secretory IgA is the predominant immunoglobulin present in gastrointestinal fluids, nasal secretions, saliva, tears and other mucous secretions of the body. IgA antibodies are important in resistance to infection of the mucosal surfaces of the body, particularly the respiratory, intestinal and urogenital tracts. IgA acts as a protective coating for the mucous surfaces against microbial adherence or initial colonization. It can also neutralize toxin activity on mucosal surfaces. Fc receptors for IgA-coated microorganisms found on monocytes and neutrophils derived from the respiratory mucosa suggest that IgA may have a role in the lung, at least, in opsonization of pathogens. Secretory IgA is also transferred via the milk, i.e., the colostrum, from a nursing mother to a newborn, which provides passive immunity to many pathogens, especially those that enter by way of the GI tract.
IgE is a immunoglobulin of about 190 kD that accounts for about 0.002% of the total serum immunoglobulins. It is produced by plasma cells below the respiratory and intestinal epithelia. The majority of IgE is bound to tissue cells, especially mast cells. If an infectious agent succeeds in penetrating the IgA barrier, it comes up against the next line of defense, the MALT (mucosa-associated lymphoid tissues) system which is managed by IgE. IgE is bound very firmly to specific IgE Fc receptors on mast cells. Contact with antigen leads to release of mediators of inflammation from the mast cells, which effectively recruits various agents of the immune response including complement, chemotactic factors for phagocytes, T cells, etc. Although a well-known manifestation of this reaction is a type of immediate hypersensitivity reaction called atopic allergy (e.g., hives, asthma, hay fever, etc.), the MALT responses act as a defense mechanism because they amplify the inflammatory response and may facilitate rejection of a pathogen.
IgD is a molecule of about 175 kd that resembles IgG in its monomeric form. IgD antibodies are found for the most part on the surfaces of B lymphocytes. The same cells may also carry IgM antibody. As noted above, it is thought that IgD and IgM function as mutually-interacting antigen receptors for control of B cell activation and suppression. Hence, IgD may have an immunoregulatory function.
In addition to opsonization, activation of complement, and ADCC, antibodies have other functions in host defense including steric hindrance, toxin neutralization, agglutination, and precipitation. With regard to steric hindrance, it is understood that antibodies combine with the surfaces of microorganisms and may block or prevent their attachment to susceptible cells or mucosal surfaces. Antibody against a viral component can block attachment of the virus to susceptible host cells and thereby reduce infectivity. Secretory IgA can block attachment of pathogens to mucosal surfaces. Toxin-neutralizing antibodies (antitoxins) can also react with a soluble bacterial toxin and block the interaction of the toxin with its specific target cell or substrate. Antibodies can also combine with the surfaces of microorganisms or soluble antigens and cause them to agglutinate or precipitate. This reduces the number of separate infectious units and makes them more readily phagocytosed because the clump of particles is larger in size. Floccules or aggregates of neutralized toxin may be removed by phagocytes.
Antibodies have been proposed for use in therapy. Animals, including humans and mice have the ability to make antibodies able to recognize (by binding to) virtually any antigenic determinant and to discriminate between similar epitopes. Not only does this provide the basis for protection against disease organisms, but it makes antibodies attractive candidates to target other types of molecules found in the body, such as receptors or other proteins present on the surface of normal cells and molecules present uniquely on the surface of cancer cells. Thus the remarkable specificity of antibodies makes them promising agents for human therapy.
Initial antibody preparations available for use, such as intravenous gammaglobulins, included animal and human antisera that were used in vivo to destroy bacteria (tetanus, pneumococcus) and neutralize virus (hepatitis A and B, rabies, cytomegalovirus, and varicella zoster) in the blood of infected individuals. Possibly the most important early application was the use and endotoxins. However, there are problems associated with the use of antibodies in human therapy because the response of the immune system to any antigen, even the simplest, is “polyclonal,” i.e., the system manufactures antibodies of a great range of structures both in their binding regions as well as in their effector regions. Polyclonal antibody treatment was also associated with unwanted side effects. In addition to the polyclonal nature of these antibody preparations, there was the risk of infection from contaminating viruses. Serum sickness, anaphylaxis, and were also considered limiting factors. Furthermore, even if one were to isolate a single antibody-secreting cell, and place it in culture, it would die out after a few generations because of the limited growth potential of all normal somatic cells.
Until the late 1970s, polyclonal antibodies obtained from the blood serum of immunized animals, provided the only source of antibodies for research or treatment of disease. Isolation of specific antibodies was essentially impossible until Kohler and Milstein discovered how to make “monoclonal antibodies” that would have a single specificity, that would all be alike due to manufacture by a single clone of plasma cells, and that could be grown indefinitely. This technique was described in a 1975 publication (Nature 256: 495-97), and Köhler and Milstein received the 1984 Nobel Prize in Medicine for their work.
The first step in Kohler and Milstein's technique for production of monoclonal antibodies involves immunizing an experimental animal with the antigen of interest. In most of their experiments, Kohler and Milstein injected a mouse with sheep red blood cells. The mouse's body initiates an immune response and begins producing antibodies specific to the antigen. The mouse's spleen is then removed and B cells producing the antibody of interest are isolated. Tumor-producing cells that have been grown in culture are then fused with the B lymphocytes using polyethylene glycol in order to produce a “hybridoma.” Only hybridomas resulting from the fusion will survive. The spleen lymphocyte has a limited life span, so any B cells that did not fuse with a myeloma will die in the culture. As well, those cells that lack the antibody-producing aspect of the B cell will not secrete the enzyme HGPRT, which is required for growth in the HAT medium. The hypoxathine-aminopterinthymidine (HAT) medium, on which the cells are grown, inhibits the pathway for nucleotide synthesis. Cells which produce HGPRT can bypass this pathway and continue to grow. By placing the fused cells in a HAT medium, the true hybridomas can be isolated (McKay, Raff, Reichardt 1981). The isolated hybridoma cells are then screened for specificity to the desired antigen. Because each hybridoma descends from one B cell, it makes copies of only one antibody. The hybridoma that produces the antibody of interest is grown in culture to produce large amounts of monoclonal antibodies, which are then isolated for further use. The technique is called somatic cell hybridization, and the resulting hybridoma (selected for both immortality and production of the specific antibody of interest) may be cultured indefinitely, i.e., it is a potenially immortal cell line.
Monoclonal antibodies are now widely used as diagnostic and research reagents. However, their introduction into human therapy has been much slower. One principal difficulty is that mouse antibodies are “seen” by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA (“human anti-mouse antibodies”). These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys.
Two approaches have been used in an attempt to reduce the problem of HAMA. The first is the production of chimeric antibodies in which the antigen-binding part (variable regions) of a mouse monoclonal antibody is fused to the effector part (constant region) of a human antibody using genetic engineering. In a second approach, rodent antibodies have been altered through a technique known as complementarity determining region (CDR) grafting or “humanization.” In this process, the antigen binding sites, which are formed by three CDRs of the heavy chain and three CDRs of the light chain, are excised from cells secreting rodent mAb and grafted into the DNA coding for the framework of the human antibody. Because only the antigen-binding site CDRs, rather than the entire variable domain of the rodent antibody are transplanted, the resulting humanized antibody (a second generation or “hyperchimeric” antibody) is reportedly less immunogenic than a first generation chimeric antibody. This process has been further improved to include changes referred to as “reshaping” (Verhoeyen, et al., “Reshaping human antibodies: grafting an anti-lysozyme activity,” 1988 Science 239: 1534-1536; Riechmann, et al., “Reshaping human antibodies for therapy,” 1988 Nature 332: 323-337; Tempest, et al., “Reshaping human monoclonal antibody to inhibit respiratory syncitial virus infection in vivo,” Bio/Technol 1991 9: 266-271), “hyperchimerization” (Queen, et al., “A humanized antibody that binds to the human interleukin 2 receptor,” 1989 Proc Natl Acad Sci USA 86: 10029-10033; Co, et al., “Humanized antibodies for antiviral therapy,” 1991 Proc Natl Acad Sci USA 88: 2869-2873; Co, et al., “Chimeric and humanized antibodies with specificity for the CD33 antigen,” 1992 J Immunol 148: 1149-1154), and “veneering” (Mark, et al., “Derivation of therapeutically active humanized and veneered anti-CD18 antibodies. In: Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). In the reshaping process on the basis of homology, the rodent variable region is compared with the consensus sequence of the protein sequence subgroup to which it belongs. Similarly, the selected human constant region accepting framework is compared with its family consensus sequence. Gussowal, et al., “Humanization of monoclonal antibodies,” 1991 Meth Enzymol 203: 99-121. The sequence analyses identify residues, which may have undergone mutation during the affinity maturation procedure and may therefore be idiosyncratic. Inclusion of the more common human residues is said to lessen immunogenicity problems by replacing human acceptor idiosyncratic residues. Further, the reshaping process is said to allow comparison of human and rodent consensus sequences to identify any systematic “species” differences. RSV19 antibodies were humanized by employing this procedure. Taylor et al., “Humanized monoclonal antibody to respiratory syncitial virus,” 1991 Lancet 337: 1411-1412; Tempest, et al., “Reshaping a human monoclonal antibody to inhibit human respiratory syncitial virus infection in vivo,” 1991 Bio/Technol 9: 266-271. Hyperchimerization is an alternative method of identifying residues outside CDR regions that are likely to be involved in the reconstitution of binding activity. In this method, the human sequences are compared with murine variable region sequences and the one with highest homology is selected as the acceptor framework. As in the reshaping procedure, the “idiosyncratic” residues are replaced by more commonly occurring human residues. The non-CDR residues that may be interacting with the CDR sequences are identified. Finally, it is determined which one of these residues is to be included in the variable region framework. Humanized antibodies against CD33 antigen were reportedly developed by this method. Co, et al., “Chimeric and humanized antibodies with specificity for the CD33 antigen,” 1992 J Immunol 148: 1149-154. See also Carter, et al., “Humanization of an anti-p185 HER2 antibody for human cancer therapy,” 1992 Proc Natl Acad Sci USA 89: 4285-4289. The displayed surface of the protein is the primary determinant of its immunogenicity. A humanized murine antibody can thus be made less immunogenic by replacing exposed residues that differ from those commonly found in human antibodies. This method of humanization is referred to as “veneering.” Appropriate replacement of the outer residues may have little or no impact on the inner domains or interdomain framework. Veneering is a two-step process. In the first step, the most homologous human variable regions are selected and compared by each single residue to the corresponding mouse variable regions. In the second step, the mouse framework residues, which differ from its human homologue, are replaced by the residues present in the human homologue. This replacement involves only those residues that are on the surface and at least partially exposed.
Nevertheless, it took more than a quarter century of research for monoclonal antibody technology and genetic engineering methods to result in the development of immunoglobulin molecules for treatment of human diseases. Indeed, it was not until the past five years that monoclonal antibodies became as an expanding class of therapeutics. See Glennie M J and van de Winkel J G, Drug Discov Today 2003 Jun. 1; 8(11): 503-10; Souriau C and Hudson P J, “Recombinant antibodies for cancer diagnosis and therapy,” 2003 Expert Opin Biol Ther. 3(2): 305-18. See also Pendley C, et al., “Immunogenicity of therapeutic monoclonal antibodies,” 2003 Curr Opin Mol Ther. 5(2): 172-9.
All the same, an average of less than one therapeutic antibody per year has been introduced to the market beginning in 1986, eleven years after the publication of monoclonal antibodies. Five murine monoclonal antibodies were introduced into human medicine over a ten year period from 1986-1995, including “muromonab-CD3” (OrthoClone OKT3®), which binds to a molecule on the surface of T cells and was launched in 1986 to prevent acute rejection of organ transplants; “edrecolomab” (PANOREX®), launched in 1995 for treatment of colorectal cancer; “odulimomab” (ANTILFA®), launched in 1997 for use in transplant rejection; and, “ibritumomab” (ZEVALIN® yiuxetan), launched in 2002 for use in non-Hodgkin's lymphoma. Additionally, one monoclonal Fab, “abciximab” (REOPRO®), was launched in 1995. It inhibits the clumping of platelets by binding the receptors on their surface that normally are linked by fibrinogen and may be helpful in preventing reclogging of the coronary arteries in patients who have undergone angioplasty. Three chimeric monoclonal antibodies were also launched: “rituximab” (RITUXAN®), in 1997, which binds to the CD20 molecule found on most B cells and is used to treat B cell lymphomas; “basiliximab” (SIMULECT®), in 1998 for transplant rejection; and “infliximab” (REMICADE®) which binds to tumor necrosis factor-alpha (TNF-a), in 1998 for treatment of as rheumatoid arthritis and Crohn's disease. Additionally, “abciximab” (REOPRO®), a 47.6 kD Fab fragment of the chimeric human-murine monoclonal antibody 7E3 that binds to the glycoprotein (GP) IIb/IIIa receptor of human platelets, was launched in 1995 as an adjunct to percutaneous coronary intervention for the prevention of cardiac ischemic complications in patients undergoing percutaneous coronary intervention. Finally, seven “humanized” monoclonals were launched from 1997-2003: “daclizumab” (ZENAPAX®) in 1997, which binds to part of the IL-2 receptor produced at the surface of activated T cells and is used to prevent acute rejection of transplanted kidneys; “palivizumab” (SYNAGIS®) in 1998 for RSV; “trastuzumab” (HERCEPTIN®) in 1998, which binds HER-2, a growth factor receptor found on breast cancers cells; “gemtuzumab” (MYLOTARG®) in 2000, which is a conjugate of a monoclonal antibody that binds CD33, a cell-surface molecule expressed-by the cancerous cells in acute myelogenous leukemia (AML) but not found on the normal stem cells needed to repopulate the bone marrow; and “alemtuzumab” (MABCAMPATH®) in 2001, which binds to CD52, a molecule found on white blood cells and has produced temporary remission of chronic lymphocytic leukemia; “adalimumab” (HUMIRA® (D2E7)), a human anti-TNF monoclonal containing human-derived heavy chain and light chain variable regions and human IgG:.kappa. constant regions was launched in 2002 for the treatment of rheumatoid arthritis; and, “omalizumab” (XOLAIR®), which binds to IgE and prevents it from binding to mast cells was approved in 2003 for the treatment of adults and adolescents over 12 years of age with moderate to severe persistent asthma who have a positive skin test or in vitro reactivity to a perennial aeroallergen and whose symptoms are inadequately controlled with inhaled corticosteroids.
Thus, protein engineering has been applied in an effort to diminish problems related to immunogenicity of administered recombinant immunoglobulin polypeptides and to try to alter antibody effector functions. However, problems remain. For example, the majority of cancer patients treated with rituximab relapse, generally within about 6-12 months, and fatal infusion reactions within 24 hours of rituximab infusion have been reported. These fatal reactions followed an infusion reaction complex that included hypoxia, pulmonary infiltrates, acute respiratory distress syndrome, myocardial infarction, ventricular fibrillation or cardiogenic shock. Acute renal failure requiring dialysis with instances of fatal outcome has also been reported in the setting of tumor lysis syndrome following treatment with rituximab, as have severe mucocutaneous reactions, some with fatal outcome. Additionally, high doses of rituximab are required for intravenous injection because the molecule is large, approximately 150 kDa, and diffusion is limited into the lymphoid tissues where many tumor cells reside.
Trastuzumab administration can result in the development of ventricular dysfunction and congestive heart failure, and the incidence and severity of cardiac dysfunction has been reported to be particularly high in patients who received trastuzumab in combination with anthracyclines and cyclophosphamide. Trastuzumab administration can also result in severe hypersensitivity reactions (including anaphylaxis), infusion reactions, and pulmonary events.
Patients receiving daclizumab immunosuppressive therapy are at increased risk for developing lymphoproliferative disorders and opportunistic infections, and it is not known whether daclizumab use will have a long-term effect on the ability of the immune system to respond to antigens first encountered during daclizumab-induced immunosuppression.
Hepatotoxicity, including severe hepatic veno-occlusive disease (VOD), has also been reported in association with the use of gemtuzumab as a single agent, as part of a combination chemotherapy regimen, and in patients without a history of liver disease or hematopoietic stem-cell transplant (HSCT). Patients who receive gemtuzumab either before or after HSCT, patients with underlying hepatic disease or abnormal liver function, and patients receiving gemtuzumab in combinations with other chemotherapy may be at increased risk for developing severe VOD. Death from liver failure and from VOD has been reported in patients who received gemtuzumab, and it has been cautioned that even careful monitoring may not identify all patients at risk or prevent the complications of hepatotoxicity.
Hepatotoxicity was also reported in patients receiving alemtuzumab. Serious and, in some rare instances fatal, pancytopenia/marrow hypoplasia, autoimmune idiopathic thrombocytopenia, and autoimmune hemolytic anemia have occurred in patients receiving alemtuzumab therapy. Alemtuzumab can also result in serious infusion reactions as well as opportunistic infections.
In patients treated with adalimumab, serious infections and sepsis, including fatalities, have been reported, as has the exacerbation of clinical symptoms and/or radiographic evidence of demyelinating disease, and patients treated with adalimumab in clinical trials had a higher incidence of lymphoma than the expected rate in the general population.
Serious adverse reactions in clinical studies with omalizumab have included malignancies and anaphylaxis, in which the observed incidence of malignancy among omalizumab-treated patients (0.5%) was numerically higher than among patients in control groups (0.2%).
Smaller immunoglobulin molecules have been constructed in an effort to overcome various problems associated with whole immunoglobulin therapy. Single chain immunoglobulin variable region fragment polypeptides (scFvs) are made of an immunoglobulin heavy chain variable domain joined via a short linker peptide to an immunoglobulin light chain variable domain. Huston et al., 1988 Proc. Natl. Acad. Sci. USA, 85: 5879-83. It has been suggested that the smaller size of scFv molecules may lead to more rapid clearance from plasma and more effective penetration into tissues than whole immunoglobulins. See, e.g., Jain, 1990 Cancer Res. 50: 814s-819s. An anti-tumor scFv was reported to show more rapid tumor penetration and more even distribution through the tumor mass than the corresponding chimeric antibody. Yokota et al., Cancer Res. 52: 3402-08 (1992).
Despite advantages that scFv molecules may have with regard to serotherapy, drawbacks to this therapeutic approach also exist. For example, rapid clearance of scFv may prevent delivery of a minimum effective dose to the target tissue. Additionally, manufacturing adequate amounts of scFv for administration to patients has been challenging due to difficulties in expression and isolation of scFv that adversely affect yields. During expression, scFv molecules lack stability and often aggregate due to pairing of variable regions from different molecules. Furthermore, production levels of scFv molecules in mammalian expression systems are reportedly low, which may limit the potential for efficient manufacturing of scFv molecules for therapy. Davis et al., 1990 J. Biol. Chem. 265: 10410-18; Traunecker et al., 1991 EMBO J. 10: 3655-59. Strategies for means to improve production have been explored, and reportedly include the addition of glycosylation sites to variable regions. See, e.g., U.S. Pat. No. 5,888,773; Jost et al., 1994 J. Biol. Chem. 269: 26267-73. Another disadvantage to the use of scFvs for therapy is the lack of effector function. An scFv that lacks the cytolytic functions, ADCC, and complement dependent-cytotoxicity may be less effective or ineffective for treating disease. Even though development of scFv technology began over 12 years ago, there are currently no scFv products approved for therapy.
Alternatively, it has been proposed that fusion of an scFv to another molecule, such as a toxin, could take advantage of the specific antigen-binding activity and the small size of an scFv to deliver the toxin to a target tissue. Chaudary et al., 1989 Nature 339: 394; Batra et al., 1991 Mol. Cell. Biol. 11: 2200. Conjugation or fusion of toxins to scFvs has thus been offered as an alternative strategy to provide potent, antigen-specific molecules, but dosing with such conjugates or chimeras can be limited by excessive and/or non-specific toxicity due to the toxin moiety of such preparations. Toxic effects may include supraphysiological elevation of liver enzymes and vascular leak syndrome, and other undesired effects. In addition, immunotoxins are themselves highly immunogenic upon administration to a host, and host antibodies generated against the immunotoxin limit potential usefulness for repeated therapeutic treatments of an individual.
Fusion proteins in which immunoglobulin constant region polypeptide sequences are present and nonimmunoglobulin sequences are substituted for the antibody variable regions have also been investigated. For example, CD4, the T cell surface protein recognized by HIV, was recombinantly fused to an immunoglobulin Fc effector domain, and an IL-2-IgG1 fusion protein reportedly effected complement-mediated lysis of IL-2 receptor-bearing cells. See Sensel et al., Chem. Immunol. 65: 129-158 (1997).
An extensive introduction as well as detailed information about all aspects of recombinant antibody technology can be found in the textbook “Recombinant Antibodies” (John Wiley & Sons, NY, 1999). A comprehensive collection of detailed antibody engineering lab Protocols can be found in R. Kontermann and S. Dubel (eds.), “The Antibody Engineering Lab Manual” (Springer Verlag, Heidelberg/NY, 2000). Diseases and disorders thought to be amenable to some type of immunoglobulin therapy include cancer and immune system disorders. Cancer includes a broad range of diseases, affecting approximately one in four individuals worldwide. Rapid and unregulated proliferation of malignant cells is a hallmark of many types of cancer, including hematological malignancies. Although patients with a hematologic malignant condition have benefited from advances in cancer therapy in the past two decades, Multani et al., 1998 J. Clin. Oncology 16: 3691-3710, and remission times have increased, most patients still relapse and succumb to their disease. Barriers to cure with cytotoxic drugs include, for example, tumor cell resistance and the high toxicity of chemotherapy, which prevents optimal dosing in many patients.
Nevertheless, patients have been treated with immunotherapeutics that target malignant cells, i.e., to antigens expressed on tumor cells. With regard to the selection of tumor cell surface antigens suitable for use as immunotherapy targets, it is preferable that such a target antigen is not expressed by normal tissues, particularly where the preservation of such tissue is important to host survival. In the case of hematologic malignancy, malignant cells express many antigens that are not expressed on the surfaces of stem cells or other essential cells. Treatment of a hematologic malignant condition using a therapeutic regimen that depletes both normal and malignant cells of hematological origin has been acceptable where regeneration of normal cells from progenitors can occur after therapy has ended. Additionally, the target antigen is desireably expressed on all or virtually all clonogenic populations of tumor cells, and it is best that expression persists despite selective pressure from immunoglobulin therapy. Strategies that employ selection of a cell surface idiotype (e.g., a particular idiotope) as a target for therapy of B cell malignancy have been limited by the outgrowth of tumor cell variants with altered surface idiotype expression, even where the antigen exhibits a high degree of tumor selectivity. Meeker et al., 1985 N. Engl. J. Med. 312: 1658-65. The selected antigen should also traffic properly after an immunoglobulin binds to it. Shedding or internalization of a cell surface target antigen after an immunoglobulin binds to the antigen may allow tumor cells to escape destruction, thus limiting the effectiveness of serotherapy. Finally, binding of an immunoglobulin to cell surface target antigens that transmit or transduce cellular activation signals may result in improved functional responses to immunotherapy in tumor cells, and can lead to growth arrest and/or apoptosis. While all of these properties are important, the triggering of apoptosis after an immunoglobulin binds to the target antigen may also be a critical factor in achieving successful serotherapy.
Antigens that have been tested as targets for serotherapy of B and T cell malignancies include Ig idiotype (Brown et al., 1989 Blood 73: 651-61), CD19 (Hekman et al., 1991 Cancer Immunol. Immunother. 32: 364-72), Vlasveld et al., 1995 Cancer Immunol. Immunother. 40: 37-47), CD20 (Press et al., 1987 Blood 69: 584-91), Maloney et al., 1997 J. Clin. Oncol. 15: 3266-74), CD21 (Scheinberg et al., 1990 J. Clin. Oncol. 8: 792-803), CD5 (Dillman et al., 1986 J. Biol. Respn. Mod. 5: 394-410), and CD52 (CAMPATH) (Pawson et al., 1997 J. Clin. Oncol. 15: 2667-72). Of these, greater benefit for therapy of B cell lymphomas has been obtained using molecules that target CD20. Other targets have been limited by biological properties of the antigen. For example, surface idiotype can be altered through somatic mutation, allowing tumor cell escape. CD5, CD21, and CD19 are rapidly internalized after monoclonal antibody binding, allowing tumor cells to escape destruction unless monoclonal antibodies are conjugated with toxin molecules. CD22 is expressed on only a subset of B cell lymphomas, thereby limiting its usefulness, while CD52 is expressed on both T cells and B cells and may therefore generate counterproductive immunosuppression by depletion.
Treatment of patients with low grade or follicular B cell lymphoma using a chimeric CD20 monoclonal antibody has been reported to induce partial or complete responses in patients. McLaughlin et al., 1996 Blood 88: 90a (abstract, suppl. 1); Maloney et al., 1997 Blood 90: 2188-95. However, as noted above, tumor relapse commonly occurs within six months to one year. Further improvements in serotherapy are needed to induce more durable responses, for example, in low grade B cell lymphoma, and to allow effective treatment of high grade lymphoma and other B cell diseases.
Another approach has been to target radioisotopes to B cell lymphomas using monoclonal antibodies specific for CD20. While the effectiveness of therapy is reportedly increased, associated toxicity from the long in vivo half-life of the radioactive antibody increases also, sometimes requiring that the patient undergo stem cell rescue. Press et al., 1993 N. Eng. J. Med. 329: 1219-1224; Kaminski et al., 1993 N. Eng. J. Med. 329: 459-65. Monoclonal antibodies to CD20 have also been cleaved with proteases to yield F(ab′)2 or Fab fragments prior to attachment of radioisotope. This has been reported to improve penetration of the radioisotope conjugate into the tumor and to shorten the in vivo half-life, thus reducing the toxicity to normal tissues. However, these molecules lack effector functions, including complement fixation and/or ADCC.
CD20 was the first human B cell lineage-specific surface molecule identified by a monoclonal antibody. It is a non-glycosylated, hydrophobic 35 kDa B cell transmembrane phosphoprotein that has both amino and carboxy ends situated in the cytoplasm. Einfeld et al., 1988 EMBO J. 7: 711-17. CD20 is expressed by all normal mature B cells, but is not expressed by precursor B cells. Natural ligands for CD20 have not been identified, and the function of CD20 in B cell biology is still incompletely understood.
Anti-CD20 monoclonal antibodies affect the viability and growth and growth of B cells. Clark et al., 1986 Proc. Natl. Acad. Sci. USA 83: 4494-98. Extensive cross-linking of CD20 can induce apoptosis in B lymphoma cell lines, Shan et al., 1998 Blood 91: 1644-52, and cross-linking of CD20 on the cell surface has been reported to increase the magnitude and enhance the kinetics of signal transduction, for example, as detected by measuring tyrosine phosphorylation of cellular substrates. Deans et al., 1993 J. Immunol. 146: 846-53. Therefore, in addition to cellular depletion by complement and ADCC mechanisms, Fc-receptor binding by CD20 monoclonal antibodies in vivo may promote apoptosis of malignant B cells by CD20 cross-linking, consistent with the theory that effectiveness of CD20 therapy of human lymphoma in a SCID mouse model may be dependent upon Fc-receptor binding by the CD20 monoclonal antibody. Funakoshi et al., 1996 J. Immunotherapy 19: 93-101. The presence of multiple membrane spanning domains in the CD20 polypeptide (Einfeld et al., 1988 EMBO J. 7: 711-17; Stamenkovic et al., 1988 J. Exp. Med. 167: 1975-80; Tedder et al., 1988 J. Immunol. 141: 4388-4394), prevent CD20 internalization after antibody binding, and this was recognized as an important feature for therapy of B cell malignancies when a murine CD20 monoclonal antibody, IF5, was injected into patients with B cell lymphoma, resulting in significant depletion of malignant cells and partial clinical responses. Press et al., 1987 Blood 69: 584-91.
Because normal mature B cells also express CD20, normal B cells are depleted by anti-CD20 antibody therapy. Reff, M. E. et al., 1994 Blood 83: 435-445. After treatment is completed, however, normal B cells can be regenerated from CD20 negative B cell precursors; therefore, patients treated with anti-CD20 therapy do not experience significant immunosuppression. Depletion of normal B cells may also be beneficial in diseases that involve inappropriate production of autoantibodies or other diseases where B cells may play a role. A chimeric monoclonal antibody specific for CD20, consisting of heavy and light chain variable regions of mouse origin fused to human IgG1 heavy chain and human kappa light chain constant regions, reportedly retained binding to CD20 and the ability to mediate ADCC and to fix complement. Liu et al., 1987 J. Immunol. 139: 3521-26. The mechanism of anti-tumor activity of rituximab, discussed above, is thought to be a combination of several activities, including ADCC, complement fixation, and triggering of signals that promote apoptosis in malignant B cells, although the large size of rituximab prevents optimal diffusion of the molecule into lymphoid tissues that contain malignant B cells, thereby limiting these anti-tumor activities. Autoimmune diseases include autoimmune thyroid diseases, which include Graves' disease and Hashimoto's thyroiditis. In the United States alone, there are about 20 million people who have some form of autoimmune thyroid disease. Autoimmune thyroid disease results from the production of autoantibodies that either stimulate the thyroid to cause hyperthyroidism (Graves' disease) or destroy the thyroid to cause hypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid is caused by autoantibodies that bind and activate the thyroid stimulating hormone (TSH) receptor. Destruction of the thyroid is caused by autoantibodies that react with other thyroid antigens. Current therapy for Graves' disease includes surgery, radioactive iodine, or antithyroid drug therapy. Radioactive iodine is widely used, since antithyroid medications have significant side effects and disease recurrence is high. Surgery is reserved for patients with large goiters or where there is a need for very rapid normalization of thyroid function. There are no therapies that target the production of autoantibodies responsible for stimulating the TSH receptor. Current therapy for Hashimoto's thyroiditis is levothyroxine sodium, and therapy is usually lifelong because of the low likelihood of remission. Suppressive therapy has been shown to shrink goiters in Hashimoto's thryoiditis, but no therapies that reduce autoantibody production to target the disease mechanism are known.
Rheumatoid arthritis (RA) is a chronic disease characterized by inflamation of the joints, leading to swelling, pain, and loss of function. RA effects an estimated 2.5 million people in the United States. RA is caused by a combination of events including an initial infection or injury, an abnormal immune response, and genetic factors. While autoreactive T cells and B cells are present in RA, the detection of high levels of antibodies that collect in the joints, called rheumatoid factor, is used in the diagnosis of RA. Current therapy for RA includes many medications for managing pain and slowing the progression of the disease. No therapy has been found that can cure the disease. Medications include nonsteroidal antiinflamatory drugs (NSAIDS), and disease modifying antirheumatic drugs (DMARDS). NSAIDS are useful in benign disease, but fail to prevent the progression to joint destruction and debility in severe RA. Both NSAIDS and DMARDS are associated with signficant side effects. Only one new DMARD, Leflunomide, has been approved in over 10 years. Leflunomide blocks production of autoantibodies, reduces inflamation, and slows progression of RA. However, this drug also causes severe side effects including nausea, diarrhea, hair loss, rash, and liver injury.
Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused by recurrent injuries to blood vessels in multiple organs, including the kidney, skin, and joints. SLE is estimated to affect over 500,000 people in the United States. In patients with SLE, a faulty interaction between T cells and B cells results in the production of autoantibodies that attack the cell nucleus. These include anti-double stranded DNA and anti-Sm antibodies. Autoantibodies that bind phospholipids are also found in about half of SLE patients, and are responsible for blood vessel damage and low blood counts. Immune complexes accumulate the kidneys, blood vessels, and joints of SLE patients, where they cause inflamation and tissue damage. No treatment for SLE has been found to cure the disease. NSAIDS and DMARDS are used for therapy depending upon the severity of the disease. Plasmapheresis with plasma exchange to remove autoantibodies can cause temporary improvement in SLE patients. There is general agreement that autoantibodies are responsible for SLE, so new therapies that deplete the B cell lineage, allowing the immune system to reset as new B cells are generated from precursors, would offer hope for long lasting benefit in SLE patients.
Sjogren's syndrome is an autoimmune disease characterized by destruction of the body's moisture-producing glands. Sjogren's syndrome is one of the most prevelant autoimmune disorders, striking up to an estimated 4 million people in the united states. About half of people stricken with Sjogren's syndrome also have a connective tissue disease, such as RA, while the other half have primary Sjogren's syndrome with no other concurrent autoimmune disease. Autoantibodies, including anti-nuclear antibodies, rheumatoid factor, anti-fodrin, and anti-muscarinic receptor are often present in patients with Sjogren's syndrome. Conventional therapy includes corticosteroids, and additional more effective therapies would be of benefit.
Immune thrombocytopenic purpura (ITP) is caused by autoantibodies that bind to blood platelets and cause their destruction. Some cases of ITP are caused by drugs, and others are associated with infection, pregnancy, or autoimmune disease such as SLE. About half of all cases are classified as “idiopathic”; meaning the cause is unknown. The treatment of ITP is determined by the severity of the symptoms. In some cases, no therapy is needed although in most cases immunosuppressive drugs, including corticosteroids or intravenous infusions of immune globulin to deplete T cells, are provided. Another treatment that usually results in an increased number of platelets is removal of the spleen, the organ that destroys antibody-coated platelets. More potent immunosuppressive drugs, including cyclosporine, cyclophosphamide, or azathioprine are used for patients with severe cases. Removal of autoantibodies by passage of patients' plasma over a Protein A column is used as a second line treatment in patients with severe disease. Additional more effective therapies are desired.
Multiple sclerosis (MS) is also an autoimmune disease. It is characterized by inflamation of the central nervous system and destruction of myelin, which insulates nerve cell fibers in the brain, spinal cord, and body. Although the cause of MS is unknown, it is widely believed that autoimmune T cells are primary contributors to the pathogenesis of the disease. However, high levels of antibodies are present in the cerebral spinal fluid of patients with MS, and some theories predict that the B cell response leading to antibody production is important for mediating the disease. No B cell depletion therapies have been studies in patients with MS, and there is no cure for MS. Current therapy is corticosteroids, which can reduce the duration and severity of attacks, but do not affect the course of MS over time. New biotechnology interferon (IFN) therapies for MS have recently been approved but additional more effectiver therapies are desired.
Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorder that is characterized by weakness of the voluntary muscle groups. MG effects about 40,000 people in the united states. MG is caused by autoantibodies that bind to acetylcholine receptors expressed at neuromuscular junctions. The autoantibodies reduce or block acetylcholine receptors, preventing the transmission of signals from nerves to muscles. There is no known cure for mg. Common treatments include immunosuppression with corticosteroids, cyclosporine, cyclophosphamide, or azathioprine. Surgical removal of the thymus is often used to blunt the autoimmune response. Plasmapheresis, used to reduce autoantibody levels in the blood, is effective in mg, but is short-lived because the production of autoantibodies continues. Plasmapheresis is usually reserved for severe muscle weakness prior to surgery. New and effective therapies would be of benefit.
Psoriasis effects approximately five million people, and is characterized by autoimmune inflammation in the skin. Psoriasis is also associated with arthritis in 30% (psoriatic arthritis). Many treatments, including steroids, uv light retenoids, vitamin d derivatives, cyclosporine, methotrexate have been used but it is also plain that psoriasis would benefit from new and effective therapies. Scieroderma is a chronic autoimmune disease of the connective tissue that is also known as systemic sclerosis. Scleroderma is characterized by an overproduction of collagen, resulting in a thickening of the skin, and approximately 300,000 people in the united states have scleroderma, which would also benefit from new and effective therapies.
There is a clear need for improved compositions and methods to treat malignacies, including B cell malignancies and disorders including autoimmnune diseases, disorders, and conditions, as well as other diseases, disorders, and conditions. The compositions and methods of the present invention described and claimed herein provide such improved compositions and methods as well as other advantages.